The use of optical detection has proved very useful in biological and chemical analytical systems. The use of optically detectable binding agents and optically detectable reactions have provided highly sensitive target detection in analytical systems. The use of a fluorescent label enables highly sensitive detection without the potential hazards of radioactive detection systems. Fluorescent detection may be effected as an array of reactions in separate reaction containers allowing for higher through-put assays. In addition, different markers emitting fluorescence at different wavelengths allow for the simultaneous measurement of multiple parameters in each assay, with each parameter measured with a different optically detectable marker with specific optical properties. Each optical marker would be characterized by a specific detectable emission wavelength. Optical detection may be effected either as in-vitro studies in which compounds bind to a solid support or as in-vivo assays wherein a labeled binding agent selectively binds to an assay target on the cell.
In optical detection assays, the use of detection in a homogenous assay mixture allows rapid detection with minimal processing steps. In such a detection assay, the sample is analyzed without purification steps. The homogenous assay mixture would thus contain the Sample material to be tested and the reagents added to the sample. At least one of the added reagents would react with the sample to form a binding site-binding agent complex to make a target optically detectable. Analyzing the homogenous assay mixture enables detection of the target without purification steps. This decreases assay error, makes the assay more rapid and saves experimentalist time. In addition, absolute target measurement (i.e. target number per unit volume) is enabled by measuring individual targets, such as cells, in a homogenous assay.
One limitation with such optical detection in a homogenous liquid is the optical background. The unbound or unreacted reagent that makes the target optically detectable is present in the reaction mixture. In addition other elements in the reaction mixture, such as components from the unpurified test sample, also cause optical interference. One method to assuage this problem is to use an optical system which directs light of a specific wavelength at the target. This light can be selected to minimize optical interference by providing illumination specifically directed to excite fluorescence from the label on the binding agent but to be outside of the wavelengths which would excite autofluorescence from the sample.
However, this will not solve the problem of optical interference from the unbound or unreacted reagent used as the binding agent to make the target optically detectable. This reagent will have the same optical properties both when bound to the target and when free in the assay mixture. The solution to this problem has been to limit the depth of field for the optical detection system. The targets commonly have a characteristic density or adhere to a solid support at a discrete location. The targets naturally concentrate at one depth with the sample container. If the container contains a density gradient media, the sample will form a band at a specific density layer. Alternatively the assay can be configured so that the targets bind to a static surface at a specific depth within the sample container. In either case the optical detection system need detect only for a limited depth of field. One example of an optical instrument that limits the depth of field is a confocal microscope. Alternatively the IMAGN 2000 from Biometric Imaging combines optical elements to provide a raster line scan of a depth of 25 nm or more within a container. See U.S. Pat. No. 5,547,849. In such a limited depth of field the amount of background measured from the unbound or unreacted reagent would be significantly less than in the total volume. This allows the target, which contains a much higher level of the optically detectable marker associated with the reagent to be detected. However, the amount of the background from the unbound or unreacted reagent is still significant. This lowers the detection threshold for the target compound or complex. In addition, the amount of reagent with associated optically detectable marker added to the assay mixture must be limited to control background. For some assays, adding a higher amount of this reagent may allow improved reaction speed or enable lower levels of detection. However, to reduce background higher levels of binding agent are precluded. A method to separate the assay reagents from the targets band to binding agents is needed.
It is the object of the invention to provide a method of optically detecting targets in a liquid sample containing unbound fluorescent reagents with lower background. This improved method should provide the ability to use a greater range of wavelengths, lower detection limits and possibly increase detection speed.
A further object of the invention is to describe an assay system for the optical detection of targets with reduced background.